CD16 (also known as FcγRIII), a low affinity receptor for the Fc portion of some IgGs known to be involved in antibody-dependent cellular cytotoxicity (ADCC), is the best-characterized membrane receptor responsible for triggering of target cell lysis by NK cells (Mandelboim et al., 1999, PNAS 96:5640-5644). Human NK cells comprise approximately 15% of all lymphocytes and are defined phenotypically by their expression of CD56 and lack of expression of CD3 (Robertson and Ritz, 1990, Blood 76:2421-2438). The majority (approximately 90%) of human NK cells express CD56 at low density (CD56dim) and FcγRIII (CD16) at a high level (Cooper et al., 2001, Trends Immunol. 22:633-640). Human FcγRIII exists as two isoforms, FcγRIIIA and FcγRIIIB, that share 96% sequence identity in their extracellular immunoglobulin-binding regions (van de Winkel and Capel, 1993, Immunol. Today 14(5):215-221).
FcγRIIIA is expressed on macrophages, mast cells, and NK cells as a transmembrane receptor. On NK cells, the alpha chain of FcγRIIIA associates with the immunoreceptor tyrosine-based activation motif (ITAM) containing FcεRI γ-chain and/or the T-cell receptor (TCR)/CD3 ζ-chain to mediate signalling (Wirthmueller et al., 1992, J. Exp. Med. 175:1381-1390). The interaction of FcγRIIIA with different combinations of homo- and hetero-dimers of the γ and ζ chains has been observed in NK cells, offering the possibility of different signalling pathways via variations of the FcγRIIIA complex in NK cells (Anderson et al., 1990, PNAS 87(6):2274-2278; Ackerly et al., 1992, Int. J. Cancer Suppl. 7:11-14).
FcγRIIIB is present on polymorphonuclear granulocytes (PMN) as a glycosyl-phosphatidylinositol (GPI)-anchored receptor (FcγRIIIB isoform), which cannot trigger tumour cell killing (van de Winkel and Capel, 1993, supra). In addition, FcγRIIIB exists as a soluble receptor in serum, and upon binding to antibodies may trigger side-effects via the formation of immune complexes in vivo.
FcγR-expressing effector cells have been shown to be involved in destroying tumour cells via ADCC. This has lead to the development of several immunotherapeutic approaches to cancer therapy, which involve the use of FcγR activities, for example, tumour specific monoclonal antibodies can mediate destruction of tumour cells by ADCC induced via binding to FcγRs, and bi-specific molecules with one specificity for tumour cells and the other specificity for either an FcγRI on granulocytes or FcγRIII on NK cells have also been developed in efforts to improve effector cell recruitment (van Spriel et al., 2000 Immunol. Today, 21:391-397).
As cellular mediators of innate immunity, NK cells are professional cell killers and, unlike T cells, tend to exist in constitutively activated states not requiring additional (pre-)stimulation. In contrast, resting cytotoxic T-cells require activation via the binding of an antigen-MHC complex and subsequent co-stimulation via CD28. Thus, amongst the immune effector cells, the NK cell is one of the more attractive candidates for use in immunotherapy, and bi-specific antibodies with specificity to CD16 (FcγRIII) and HER-2/neu or CD16 and CD30 have been developed (Arndt et al., 1999 Blood 94:2562-2568; McCall et al., 2001 J. Immunol. 166:6112-6117).
The majority of anti-CD16 antibodies that have been produced to date, are unable to distinguish between the two CD16 isoforms, FcγRIIIA and FcγRIIIB, with the exceptions being monoclonal antibody (MAb) 1D3, which only binds to the form of CD16 expressed on neutrophils i.e. FcγRIIIB, and the monoclonal antibodies which recognise different allotypic forms of FcγRIIIB (Ory et al. 1989, J. Clin. Invest. 83:1676-81; Huizinga et al. 1990 Blood 75:213-7; Tamm & Schmidt 1996, J. Immunol. 1996 157:1576-1581). However, to date, there have been no examples of molecules that bind specifically to FcγRIIIA, the form expressed on NK cells, and not to FcγRIIIB.
An antibody specific for FcγRIIIA, however, would be particularly useful as a component of a bi- or multispecific binding molecule that is directed against disease-associated cells, as it would mainly recruit NK cells, and would not be bound by circulating soluble FcγRIIIB or diverted from NK cell binding by binding to neutrophils or activated eosinophils. To efficiently mediate NK-cell killing, the FcγRIIIA-specific antibody must not only bind NK cells but also activate them upon binding.